1. Field of the Invention
This invention relates to compositions comprising, as the main ingredient, allogenic activated-CD4+ cells which are derived from a different person, and a method for producing and dispensing the same. More particularly, this invention relates to compositions effective in preventing recidivation of a tumor and remedying various infections such as viral infections, and autoimmune diseases such as phagocytosis.
2. Description of the Related Art
Lymphocytes take part in the immune system concerned in biophylaxis. In particular, T-lymphocytes are one kind of important cells playing a major role in cellular immunity. The lymphocytes are sorted in accordance with the reactivity of the monoclonal antibody. For example, the T-lymphocytes having the reactivity to anti-CD3 antibodies are regarded as CD3+ cells. There have been many studies in the relation between antigens manifested in the lymphocytes and its function. The T-lymphocyte manifests not only the CD3 antigen, but also other antigens of various kinds at the same time. The lymphocytes manifesting CD4 antigens (hereinafter referred to as xe2x80x9cCD4+ cellsxe2x80x9d) have been considered to have a function of activating lymphocytes manifesting CD8 antigens (hereinafter referred to as xe2x80x9cCD8+ cellsxe2x80x9d). The CD8+ cell has intense cytotoxic activity. However, the T-lymphocytes have various functions, and thus, they have been generally considered to be difficult to sort only in accordance with the manifesting superficial antigen. It has been reported that the CD4+ cells have the cytotoxic activity in, for example, xe2x80x9cCytotoxic Activity of CD4+ T-cells against Autologous Tumor Cellsxe2x80x9d by Y. Konishi, T. Sekine, T. Takayama, M. Fujii, and T. Tanaka, Jpn J. Cancer, Res., Vol.86, pp.854-860 (1995).
T. Sekine, one of the inventors of the present invention, disclosed that the lymphocytes can be proliferated by solid-phase anti-CD3 antibody and interleukin-2, and autologous lymphocytes thus proliferated have an antitumor effect (Japanese Patent Application Public Disclosure No. HEI 3-80076(A)). There have been many other reports that the lymphocytes derived from peripheral blood and so on can be proliferated by the anti-CD3 antibody and interleukin-2, and the autologous lymphocytes have antitumor activity.
Ito et al., ones of the inventors of the present invention, reported that the autologous lymphocytes proliferated from the anti-CD3 antibody and interleukin-2 are effective in the treatment of viral infections of a sufferer from congenital immunodeficiency (xe2x80x9cCourse of Medicinexe2x80x9d by Kiminari Ito and Teruaki Sekine, Vol.181, No.6, pp.426-427 (1997)).
A bone-marrow transplant is performed when leukocyte blood types (hereinafter called xe2x80x9cHLAxe2x80x9d for short) of both donor and recipient agree therewith. However, because various types of HLA are in existence, it is exceedingly rare for the HLA types of the donor and recipient to agree with each other. Accordingly, when the major components of HLA of the donor are in agreement with those of the recipient, the bone-marrow transplant is generally carried out. Failure of complete agreement of the HLA types sometimes causes the graft-versus-host disease (hereinafter called xe2x80x9cGVHDxe2x80x9d for short) which bring about severe symptoms. For the purpose of remedying the disease, an immunosuppressant has been used. A patient developing the disease may possibly recuperate from GVHD with administration of the immunosuppressant, as a result of which the patient will infrequently manifest viral infections attributable to cytomegalovirus or Epstein-Barr virus and eventually be brought death in most cases.
Elizabeth et al. reported that CD8+ cells specific to cytomegalovirus are derived from lymphocytes of a bone-marrow donor for the purpose of preventing and treating the viral infections caused in the immunosuppressive conditions, so that the viral infections attributable to cytomegalovirus which the recipient is infected with can be treated. (Elizabeth A. Walter, M.D. et al., N. Engl. J. of Med., Vol.333, pp.1038-1044 (1995))
In the actual clinical cases, allogenic lymphocytes prepared by a pheresis operation have been used for the purpose of treating diseases such as leukemia. This treatment is called xe2x80x9cdonor leukocyte transfusionxe2x80x9d (hereinafter called xe2x80x9cDLTxe2x80x9d for short). DLT shows a beneficial effect, but it has been found that 50% to 80% of acute GVHD or 20% of fatal GVHD emerged in the recipient (H. J. Kolb et al., xe2x80x9cBloodxe2x80x9d, vol.86, pp.2041-2050 (1995); S. Slavin et al., xe2x80x9cExp. Hematolxe2x80x9d, Vol. 23, pp.1553-1562 (1995)). To make matters worse, it takes several hours to perform the pheresis operation, and the operation imposes a severe burden on the donor.
Giralt et al. reported about DLT using leukocytes excluding CD8+ cells (Giralt et al., xe2x80x9cBloodxe2x80x9d, vol.86, pp.4337-4343 (1995). Ritz et al. reported about DLT using CD4+ cells prepared by a pheresis operation (Claret E. J. et al., xe2x80x9cJ. Clin. Investxe2x80x9d, vol.100, No.4, pp.588-866 (1997)). In either case, it was reported that the effect of GVL (graft-versus-leukemia, a slight case of which favorably reacts on a patient) was derived without causing GVHD, but it is feeble.
The allogenic lymphocytes specific to the antigen are efficacious against the viral infections without causing GVHD. However, the effective spectrum thereof is confined only to the specified viruses. Consequently, the necessity of deriving various types of antigen-specific lymphocytes to cope with various kinds of viral infections arises. In particular, when infections caused by unexpected pathogens such as viruses are developed, the antigen-specific allogenic lymphocytes cannot be used because it takes much time to derive the antigen-specific lymphocytes.
Furthermore, it is difficult to derive the antigen-specific lymphocytes effective for infections in which their pathogenic germs cannot be identified. Thus, there has been a need for allogenic lymphocytes which have wider effective spectrum or are not restricted to the specific effective spectrum.
Administration of the allogenic lymphocytes such as DLT can be expected to produce the intended beneficial curative effect, e.g. antitumor effect, but it consequently increases a possibility of causing serious GVHD with greater frequency. DLT using simply sorted CD4+ cells does not produce GVHD, but has little curative effect. Moreover, DLT necessitates gathering of leukocytes from a donor by the pheresis operation which imposes a severe physical burden on the donor and requires special facilities for fulfilling the operation. Particularly, when the donor is an infant, it is difficult to extract the leukocytes in large quantities therefrom. It has been desired to devise medicaments or preparations which have wide applicability and high efficiency, and besides, can be prepared lessening a burden on the donor without using special facilities and applied clinically without causing GVHD.
An object of the present invention is to provide compositions of lymphocytes applicable for remedying various diseases without causing serious GVHD, which have an excellent curative effect of restraining recidivation of a tumor, and remedying viral infections and autommune diseases and can be widely applied to immunological diseases and bring about marked curative effects in performing a specific treatment.
Another object of the invention is to provide a medical kit for preparing, with high efficiency, the compositions of lymphocytes applicable for remedying various diseases without causing serious GVHD, which have an excellent curative effect of restraining recidivation of a tumor, and remedying viral infections and autommune diseases and can be widely applied to immunological diseases.
Still another object of the invention is to provide a method for preparing, with high efficiency, the compositions of lymphocytes applicable for remedying various diseases without causing serious GVHD, which have an excellent curative effect of restraining recidivation of a tumor, and remedying viral infections and autommune diseases and can be widely applied to immunological diseases.
To attain the objects described above according to this invention, there is provided compositions or medicaments prepared by separating lymphocyte cells or CD4+ cells from a small quantity of peripheral blood of a donor, and proliferating the cells thus prepared, to obtain allogenic activated-CD4+ cells to be contained therein as the main ingredient.
The medicament according to this invention contains, as the main ingredient, the allogenic activated-CD4+ cells and has an excellent curative effect of restraining recidivation of a tumor, and remedying viral infections and autommune diseases without causing serious GVHD.
The allogenic activated-CD4+ cells may be derived from an organ donor or bone-marrow donor.
The medicament according to this invention may contain 90% of allogenic activated-CD4+ cells relative to the total amount of cells in the medicament.
The medicament according to this invention may contain CD8+ cells relative to the total amount of cells in the medicament.
A method for producing the medicaments according to this invention, which contain, as the main ingredient, the allogenic activated-CD4+ cells, comprises separating CD4+ cells from peripheral blood of a donor, and proliferating the cells thus prepared in the presence of a proliferation activator, to obtain allogenic activated-CD4+ cells to be contained therein as the main ingredient.
Another method for producing the medicaments according to this invention, which contain, as the main ingredient, the allogenic activated-CD4+ cells, comprises removing CD8+ cells from peripheral blood of a donor by using anti-CD8+ antibodies, and proliferating the cells thus prepared in the presence of a proliferation activator, to obtain allogenic activated-CD4+ cells to be contained therein as the main ingredient.
Still another method for producing the medicaments according to this invention, which contain, as the main ingredient, the allogenic activated-CD4+ cells, comprises proliferating allogenic lymphocyte cells in the presence of a proliferation activator, and separating activated-CD4+ cells from the allogenic lymphocyte cells by using anti-CD4+ antibodies, to obtain allogenic activated-CD4+ cells to be contained therein as the main ingredient.
A further method for producing the medicaments according to this invention, which contain, as the main ingredient, the allogenic activated-CD8+ cells, comprises proliferating allogenic lymphocyte cells in the presence of a proliferation activator, and separating activated-CD8+ cells from the allogenic lymphocyte cells by using anti-CD8+ antibodies, to obtain allogenic activated-CD4+ cells to be contained therein as the main ingredient.
In any method, interleukin-2 and/or anti-CD3 antibodies may be used as the proliferation activator.
The allogenic activated-CD4+ cells may be derived from an organ donor or bone-marrow donor.
The allogenic activated-CD4+ cell in the medicament according to the invention is defined as a CD4+ cell derived from a different person other than a recipient and activated with a proliferating activator having a proliferation activity. As the proliferating activator, interleukin-2, anti-CD3 antibodies and the like can be enumerated.
The allogenic activated-CD4+ cells may be derived. from any different person. However, in a case that a patient have already been subjected to bone-marrow or organ transplant surgery, the allogenic activated-CD4+ cells are preferably derived from the bone-marrow and/or organ donor from a medical point of view to lessen the risk of GVHD possibly caused by CD8+ cells or the like, prevent recidivation of a tumor, and remedy various infections such as viral infections, and autoimmune diseases such as phagocytosis.
In particular, for the purpose of avoiding the risk of GVHD caused by CD8+ cells or the like, it is preferable to contain 90% or more activated-CD4+ cells in the medicaments of the invention, which contain, as the main ingredient, allogenic activated-CD4+ cells, relative to the total amount of the cells in the medicament. More preferably, the medicaments of the invention have 95% or more activated-CD4+ cells contained therein relative to the total amount of the cells. That is, the medicament containing, as the main ingredient, allogenic activated-CD4+ cells, is desired to not only increase the content of activated-CD4+ cells, but also contain CD8+ cells as little as possible. To put it concretely, it is desirable to reduce the content of CD8+ cells in the medicament to 10% or less, preferably, 5% or less, more preferably, 1% or less relative to the total of cells in the medicament.
The allogenic activated-CD4+ cells contained in the medicament of the invention may assume any formation.
For example, the allogenic activated-CD4+ cells suspended in an adequate solution may be used. The solution containing the allogenic activated-CD4+ cells can desirably be used as an injection or drip-feed solution. Especially, an injection or drip-feed solution, which is prepared by suspending the allogenic activated-CD4+ cells in physiological saline and so on containing 0.01% to 5% of human seralbumin, is more suitable, but this should not be understood as a limitation.
The allogenic activated-CD4+ cells or the preparations containing them, which are produced by the method of this invention, may be frozen and kept in their frozen state so as to be used for remedying or preventing various disease.
When the medicaments according to the invention can desirably be administered to a patient by an intravenous drip, venoclysis, arterial injection, local injection and so on. The desirable dosage of the medical solution varies in accordance with the way or place of the administration thereof. However, it is commonly desirable to administer 50 to 500 ml of the medical solution containing the allogenic activated-CD4+ cells in the aforesaid ratio to the patient. It is preferable that the medical solution is administered one time a day to one time a month. In any event, at least one administration of the medicament containing the allogenic activated-CD4+ cells should be made.
The dosage of the allogenic activated-CD4+ cells contained, as the main ingredient, in the medicament of the invention, may be arbitrarily decided in accordance with the condition of the patient and/or the clinical procedure. In general, 1xc3x97102 to 1xc3x97109 units of allogenic activated-CD4+ cells per kilogram of patient""s weight may be used. It is more desirable to determine the dosage of the allogenic activated-CD4+ cells to 1xc3x97104 to 1xc3x97108 units in the light of the curative effect of the preparations.
The allogenic activated-CD4+ cells contained in the preparations according to this invention may contain various genetic factors, or be prepared by being deprived of or mutating intrinsic genetic factors.
Also, the medicaments or preparations according to this invention can be effectively used for preventing or treating various infections in the conditions of immunodeficiency caused by human immunological deficient virus as the result of using the activated-CD4+ cells short of necessary cofactors taking part in virus infections of human immunodeficiency.
The medicaments or preparations according to this invention can be used regardless of whether or not a donor and a recipient agree in HLA with each other.
The medicaments or preparations of the invention can be applied to patients who are subjected to a bone-marrow transplant or the like, and contracting various infectious diseases, cancer, immunodeficiency, autommune diseases or allergosis.
The infectious diseases include viral infections, bacteriosis, mycetogenic infection, protozoiasis infection, clamydial infection, mycoplasma infection, and the like. The viral infections are attributable to cytomegalovirus and Epstein-Barr virus and other possible viruses. That is, the medicaments of the invention can be applied for preventing and treating various viral infections including those infected by herpes simplex virus, herpesvirus such as varicella zoster virus, human leukosis virus, various retrovirus such as human immunological deficient virus. The microbism on which the medicaments of the invention are effective is typified by the infections attributable to pseudomonas aeruginosa, methicillin-resistant staphylococcus aureus, or the like. In any event, the medicaments and preparations according to this invention are effective on any infections attributable to germs pathogenically affecting humans. Especially, the present invention can be applied for remedying and preventing the infections caused by pathogenic germs which have been unknown or cannot be identified or diseases accompanying the infections.
The cancerous diseases to which the medicaments of the invention can be applied are typified by leukemia. However, the medicaments of the present invention is not applied only to this disease, but effective in remedying various solid carcinoma. Namely, the medicaments and preparations of the invention are effective in not only preventing the recidivation of a tumor, but also remedying the tumor.
The immunodeficiency on which the medicaments of the invention is effective is roughly divided into congenital immunodeficiency and acquired immunodeficiency. The congenital immunodeficiency includes severe combined immunodeficiency, Wiscott-Aldrich syndrome, adenosine deaminase deficiency, and purine nucleotide-phosphorylase deficiency. However, the prescription with the medicaments and preparations of the invention is not limited only to the immunological deficient diseases enumerated herein. The acquired immunodeficiency includes secondary immunodeficiency caused by use of carcinostatic, immunosuppressive agent, steroid or the like. The medicaments of the invention are not only effective on these diseases, but also applicable to patients short or lack of immunological competence against specific viruses.
The autommune diseases are typified by systemic lupus erythematosus, rheumatoid arthritis, Sjogren syndrome, myasthenia gravis, pernicious anemia, Hashimoto disease, and so on. However, the medicaments of the invention is not applicable only to these diseases.
The allergosis includes bronchial asthma, cryptomeria pollinosis, and urticaria. However, the medicaments according to this invention are not applicable only to these allergic diseases, but can be effectively used for remedying or mitigating these and other allergic diseases.
The method for producing medicaments or preparations which contain, as the main ingredient, allogenic activated-CD4+ cells according to the present invention will be described hereinafter.
The allogenic activated-CD4+ cells to be contained therein as the main ingredient are obtained by separating lymphocyte cells from a small quantity of peripheral blood of a donor, and proliferating the cells thus prepared in a test tube. In a different method, the allogenic activated-CD4+ cells may be obtained by separating lymphocyte cells from a small quantity of peripheral blood of a donor, further separating CD4+ cells, and proliferating the cells thus prepared in a test tube in a similar manner.
The extraction of allogenic lymphocyte or CD4+ cells from the donor may be performed any way, for example, by blood collection, pheresis, or other possible operations. Taking into consideration a physical burden imposed on the donor, the method of extracting the lymphocyte cells or CD4+ cells from the peripheral blood of the donor, which can be carried out with ease, is recommendable.
It is desirable to draw blood from the vein of the donor, and add heparin or citric acid to the blood thus drawn to prevent blood coagulation. The blood of the order of 0.01 ml to 100 ml is generally drawn in one blood extraction operation, but the amount of the blood to be drawn is not limited in the invention. Taking into consideration the physical burden of the donor, labors involved in collecting the blood, and troublesome operations for separating the lymphocyte cells, it is desirable to drawn the blood by 5 ml to 10 ml, preferably 10 ml to 20 ml in one blood extraction operation.
The operation for separating the lymphocyte cells from the blood drawn in the aforementioned manner may be accomplished by a known method for separating lymphocyte cells such as a discontinuous density gradient centrifugation method which is performed by using sucrose or lymphocyte separating agents on the market.
The proliferation of the lymphocyte cells or CD4+ cells in the invention may be effected by a known lymphocyte cultivating method. The cultivation method as disclosed in Japanese Patent Application Public Disclosure No. HEI 3-80076(A) is applicable in this invention by way of example, but should not be construed as a limitation.
In view of the efficiency of proliferating the lymphocyte cells or CD4+ cells, the cultivation of the desired cells is preferably effected in the presence of interleukin-2 used as the proliferating activator. It is more desirable to effect the cultivation in the presence of the interleukin-2 and anti-CD3 antibodies. As one measure, the cultivation may be carried into practice by allowing lymphocyte cells floating in a culture medium solution containing interleukin-2 and putting the solution into a culture flask in which the anti-CD3 antibodies assume their solid phase. Also, various kinds of mitogens may be used as required to activate the cultivation of the CD4+ cells.
The type of the anti-CD3 antibodies used in the invention is not limited to a specific antibody, as far as the antibody makes for proliferation and activation of the desired lymphocyte cells. The anti-CD3 antibodies used for stimulating the lymphocyte cells are possibly yielded in organisms or organic cells by use of refined CD3 molecules. However, from the viewpoint of stability and cost, it is advisable to use OKT-3 antibodies marketed by Ortho Pharmaceutical Corp.
To efficiently proliferate the lymphocyte cells with ease, the anti-CD3 antibodies are desired to be solidified. As an instrument for solidifying the antibodies, there may be used a culture flask or vessel of glass, polyurethane, polyolefine, polystyrene or the like. Sterilized cell-culture flasks made of plastic of various sizes have been on the market, and therefore, can be chosen at pleasure.
The solidification of the aforenoted anti-CD3 antibodies is accomplished by pouring the diluent solution of anti-CD3 antibodies into the instrument for solidifying and permitting it to stand at 4xc2x0 C. to 37xc2x0 C. for 2 to 24 hours. For the solidification of the anti-CD3 antibodies, the anti-CD3 antibodies are desired to be diluted in a physiological buffer solution such as a sterilized dulbecco phosphate buffer solution to the concentration of 1 to 30 xcexcg/ml.
After solidifying, the antibodies can be preserved in a cold room or refrigerator (at 4xc2x0 C.) until using. When, the solution is removed, and if need arises, the solidified antibodies may be rinsed with the physiological buffer solution such as the dulbecco phosphate buffer solution at a normal temperature.
It is further desirable for practicing the invention to use interleukin-2 with the culture medium solution to increase the efficiency of proliferating the lymphocyte cells. The interleukin-2 is desirably used at concentration of 1 to 2000 U/ml in the culture. medium solution. The interleukin-2 on the market may be used. The interleukin-2 is used in the state dissolved in water, physiological saline, dulbecco phosphate buffer solution or a culture solution for cultivating cells, such as RPMI-1640, DMEM, IMDM, and AIM-V, which have been widely used in general. The interleukin-2 once dissolved in the culture solution is desired to be kept cold during storage to be prevented from being degraded in activity.
As the culture medium solution for cultivating the desired cells, there may be used a culture medium derived from a living organism or a culture medium composed by mixing amino acid, vitamins, nucleic acid base and so on with equilibrium salt solution, but these should be understood as limitative as far as the culture medium applied to the invention is suitable for cultivating the cells. For example, as the culture medium, RPMI-1640, AIM-V, DMEM, IMDM or the like are preferable. In particular, the culture medium of RPMI-1640 is most recommendable. The culture medium with addition of normal human serum excels in proliferating effect and can be preferably used. These culture medium applicable to the invention have been on the market.
The cultivation of the desired cells may be fulfilled by a common cell-cultivating method. For example, it can be carried out in a CO2-incubator at a CO2-concentration of 1% to 10%, preferably 5%, at a temperature of 30xc2x0 C. to 40xc2x0 C., preferably 37xc2x0 C. in particular.
The number of days which the cultivation takes is not specifically restricted, but it is desirable to allow 2 to 20 days, preferably 3 to 7 days, for the cultivating process, so as to transmit the stimulative information of the anti-CD3 antibodies to the cells. Within the period for the cultivation, it is best to observe the conditions of the cells under a microscope and take count of the number of cells so as to suitably adjust the amount of the culture medium solution by adding the solution. The proliferation of the cells does not appreciably take place within 1 to 2 days after commencement of the cultivation, but is generally observed about 3 days after the commencement. When the cells are satisfactorily proliferated, the color of the culture medium solution will be changed from orange to yellow. The loadings of the culture medium solution supplementarily added is preferably about 0.1 to 5 times the culture solution initially given. It is better to supplementarily add the culture solution every 1 to 7 days so as not to degrade the culture solution and decrease the activity of the interleukin-2.
The cultivation may possibly be continued without receiving stimulation from the anti-CD3 antibodies after cultivating the lymphocyte cells in the presence of the anti-CD3 antibodies. That is, the cultivation can be continued with an instrument having no anti-CD3 antibodies solidified, such as a cell-culture flask, a roller bottle and a gas-permeable bag for cultivation, until the medicaments are administered to a subject to be treated. It is desirable to carry out the cultivation of the lymphocyte cells under the conditions noted above in the same manner as that using the anti-CD3 antibodies except that the cells do not receive the stimulation from the solidified anti-CD3 antibodies. Timely use of serum-free culture medium in the cultivation provides advantageous high workability, economy, and safety.
When clinically using the medicaments or preparations according to this invention, it is desired to separate the CD4+ cells serving as the main ingredient for the medicaments, and simultaneously, remove CD8+ cells and CD56+ cells which can be expected to cause GVHD to obtain the CD4+ cells of high purity.
The process for separating the CD4+ cells in the invention may be performed before or after the proliferating and activating processes.
The separation of the CD4+ cells may be effected by a method of positively gathering the CD4+ cells and/or a method of removing CD8+ cells and/or CD56+ cells.
Thus, this invention does not contemplate imposing any limitation on the method of separating the CD4+ cells, CD8+ cells and/or CD56+ cells. For example, the CD4+ cells, CD8+ cells and/or CD56+ cells may be used in the state solidified on (bonded with) the surfaces of magnetic beads.
For example, the method of positively separating the CD4+ cells per se are effected by solidifying on or bonding with the surfaces of magnetic beads the anti-CD4 antibodies. As the magnetic beads applied hereto, dynabeads M-450 CD4 antibodies (anti-CD4 antibodies marked by the magnetic beads) such as DB11116 placed on the market by an importer, Veritas Corporation.
The separation of CD4+ cells can be put into practice by a commonly known separation method. Taking the efficiency of separation into consideration, it is desirable to determine the number of magnetic beads for dynabeads M-450 CD4 antibodies to 0.1 to 100 times, preferably 0.1 to 10 times, more preferably 0.5 to 5 times, that of CD4+ cells to be separated. The CD4+ cells to be separated are desired to be reacted with the dynabeads M-450 CD4 antibodies at a temperature within 4xc2x0 C. to 40xc2x0 C., taking into consideration the efficiency of reaction and the safety of cells from damage resulting from the reaction. The quantity of the cells subjected to the reaction may desirably be determined within the range of 0.5 to 1000 m/l, preferably 0.5 to 100 ml, more preferably 1 to 10 ml, taking into consideration the handling during separation and aseptic procedures. As on example of a magnet for use in separating CD4+ cells, which is by no means restricted to a specific type, Magnet MPC-1 (DB12001 imported and marketed by Veritas Corportion) may be used. The cultivation of the cells is desired to be carried on a magnetic plate.
The separation method for separation of CD4+ cells by removing CD8+ cells or CD56+ cells may be carried out by use of the anti-CD8 antibodies or the anti-CD56 antibodies solidified (bonded) on the magnetic beads. The number of the anti-CD8 antibodies or CD56 antibodies marked by the magnetic beads is desirably determined to 1 to 1000 times, preferably 1 to 200 times, more preferably 1 to 50 times, that of CD8+ cells or CD56 cells to be removed, taking into consideration of the efficiency of separating CD4+ cells.
The temperature at which CD8+ cells or CD56+ cells to be removed for separating CD4+ cells are reacted with the anti-CD8 antibodies or CD56 antibodies marked by the magnetic beads is desired to be determined to a range within 4xc2x0 C. to 40xc2x0 C., taking into consideration the efficiency of reaction and the safety of cells from damage resulting from the reaction.
The quantity of the cells subjected to the reaction to remove CD8+ cells or CD56+ cells may desirably be determined within the range of 0.5 to 1000 ml, preferably 0.5 to 100 ml, more preferably 1 to 10 ml, taking into consideration the handling during separation and aseptic procedures. Similarly to the method of separating CD4+ cells, as on example of a magnet for use in removing the CD8+ cells or CD56+ cells, which is by no means restricted to a specific type, Magnet MPC-1 (DB12001 imported and sold by Veritas Corportion) may be used.
To increase the purity of CD4+ cells to be obtained, the methods for separating CD4+ cells as described above may desirably be adopted separately or in combination. It is expediment to first remove CD8+ cells and CD56+ cells, and thereafter, positively separate the desired CD4+ cells.
As other expedient methods, a panning technique, rosetto technique or column technique may be adopted for separating CD4+ cells. Namely, the separation method is not specifically limited in the present invention, and may of course be of any one.
As described above, the allogenic activated-CD4+ cells according to this invention can be prepared by suitably using one or more solidified carriers of anti-CD4 antibodies, anti-CD8 antibodies and anti-CD56 antibodies, interleukin-2, and anti-CD3 antibodies in combination.
The medicaments of the present invention can be clinically prepared with ease by independently using such reagents as noted above, or providing a medical kit having two or more component ingredients constituting the medicaments of the invention so as to use them in combination.
For example, the medical kit of the invention may be provided by preparing the component ingredients such as the aforesaid solidified carriers of anti-CD4 antibodies, anti-CD8 antibodies and anti-CD56 antibodies, interleukin-2, and anti-CD3 antibodies as each of the reagents of the medicaments of the invention or combined reagents composed of two or more ingredients. By conveniently using the medical kit of the invention, the desired medicaments of the invention can easily be prepared in use.